| Objectives1.The possible regulation mechanism of Piezo1 on the occurrence and development of liver cancer is investigated to provide a new therapeutic target for liver cancer.2.To explore the possible mechanism and effective regulatory components of the inhibition of liver cancer by the medicated serum of Jianpi Huayu decoction(JPHY)and doxorubicin(DOX),so as to provide theoretical basis for the treatment of liver cancer by JPHY.MethodsI.Mechanism of Piezo1-mediated autophagy inhibition of hepatocellular carcinoma cell growth through PI3K/AKT/mTOR signaling pathway1.Bioinformatics analysis:Pan-cancer expression analysis of Piezo1 was performed on the Gene Expression Profiling Interactive Analysis(GEPIA)database.The Cancer Genome Atlas(TCGA)database and clinical data were used to analyze the difference in mRNA expression of Piezo1 in liver cancer tissues and normal tissues,TCGA database and gene expression synthesis(Gene Expression Omnibus,GEO)The database predicts the impact of Piezo1 on the overall survival(OS)and disease-free survival(DFS)of liver cancer patients.Gene Set Enrichment Analysis(GSEA)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to predict the molecular mechanism of Piezo1 affecting the growth of liver cancer.2.Clinical sample verification:Real-time quantitative PCR(RT-qPCR)and Western Blot were used to detect the mRNA expression level of Piezo1 in liver cancer and adjacent tissues,and the Kaplan-Meier method was used to analyze the prognosis of Piezo1 and liver cancer;Chi-square test was used to analyze the clinical correlation between Piezo1 and liver cancer patients.3.In vitro experiments:Yoda1 detects the calcium flow level of Hep3B and MHCC97H cells;constructs transient Hep3B and MHCC97H liver cancer cell lines through siPiezo1,and detects the effect of Piezo1 expression on the proliferation,invasion and migration of liver cancer.Monodansylcadaverine(MDC)was used to detect the effect of siPiezol on the autophagy of liver cancer cells;Western Blot was used to detect the protein expression level of the PI3K/AKT/mTOR pathway by inhibiting or silencing Piezo1 expression.4.Silencing Piezo1,adding autophagy inhibitor 3-MA,and verifying the effects of siPiezol and 3-MA on cell autophagy and apoptosis by clone formation assay,Western Blot.Silencing Piezo1,adding autophagy promoter Rapa,and verifying the effects of siPiezol and Rapa on cell autophagy and cell apoptosis by clone formation assay,Western Blot.5.In vivo experiments:In vitro induction of hepatocellular carcinoma by DEN to explore the effect of Piezo1 on hepatocarcinogenesis and development;MHCC97H cells transfected with siPiezol were constructed as subcutaneous tumors in nude mice to verify the effect of Piezo1 in transplanted tumors on the growth of MHCC97H cells in vivo.II.Potential mechanism of synergistic regulation of Piezo1 by JPHY drug serum and DOX and screening of effective active ingredients of JPHY1.CCK8,colony formation assay,scratch test and Transwell test were used to detect the effect of JPHY combined with DOX on inhibiting the proliferation,invasion,migration and apoptosis of HCC cells.2.Based on the systemic pharmacological approach to collect the target of DOX and the target of JPHY,to find the pathway of the joint action of DOX and JPHY.3.in vitro experiments:the cells were interfered by JPHY and DOX,and the protein expression level of the PI3K/AKT/mTOR pathway and autophagy-related protein level were detected by Western Blot.The autophagy level was detected by MDC.4.To construct a xenograft tumor mouse model to verify the effects of JPHY and DOX and the combination group on autophagy and apoptosis of transplanted tumors in mice.5.To explore the active ingredients of JPHY by mass spectrometry analysis and drug database.6.To obtain a mouse-derived Piezo1 protein crystal model from the database,to screen the effective active components of JPHY to modulate Piezo1 by molecular docking,to predict its binding ability,and to explore the regulatory mechanism of JPHY.ResultsI.Mechanism of Piezo1-mediated autophagy inhibition of hepatocellular carcinoma cell growth through PI3K/AKT/mTOR signaling pathway1.Pan-cancer expression analysis of Piezo1 in the GEPIA database revealed that Piezo1 expression was up-regulated in most tumors.Analysis of TCGA database and GEO database and clinical samples showed that Piezo1 expression in hepatocellular carcinoma tissues was higher than that in paraneoplastic tissues,and the difference were significant(P<0.05).The results of Kaplan Meier survival analysis showed that patients in the high Piezo1 expression group had worse OS and DFS than those in the low expression group(Log-rank P<0.05).Meanwhile,clinical samples likewise validated that high Piezo1 expression was associated with lower OS and DFS.2.Western Blot assay revealed that Piezo1 protein expression was significantly different in all six pairs of cancer and para-cancer tissues(P<0.05),and Piezo1 protein expression was significantly higher in cancer tissues than in para-cancer tissues.Immunohistochemical detection revealed that Piezo1 was highly expressed in cancer tissues and low expressed in para-cancer tissues.3.Western Blot detection of one normal hepatocyte line(LO2)and four hepatocellular carcinoma cell lines(MHCC97H,Huh7,HepG2,Hep3B)revealed that Piezo1 expression in hepatocellular carcinoma cell lines was significantly higher than that in tumor its cell lines.Among them,MHCC97H and Hep3B cells had the highest Piezo1 expression.4.The change of Ca2+ influx in the HCC cell line,measured by adding Piezo1 agonist Yoda 1,showed that the intracellular Ca2+ concentration in both Hep 3B and MHCC97H cell lines was significantly increased in the presence of Yoda 1(P<0.01,P<0.01).5.Screening for specific siPiezol,siPiezol inhibited the proliferation,invasion and migration of HCC cells,while siPiezol induced autophagy in HCC cells.6.The LIHC dataset of TCGA was subjected to enrichment analysis,and GESA enrichment analysis showed that the expression of Piezo1 was associated with the mTOR signaling pathway,and KEGG enrichment analysis showed that the expression of Piezo1 was associated with the PI3K/AKT signaling pathway.7.In Hep3B cells,PI3K,p-AKT,and p-mTOR protein expression levels were significantly decreased after the addition of GsMTx4 compared with the control group(P<0.01);compared with the siCtrl group,PI3K protein expression levels were decreased after transfection with siPiezo1(P<0.05),p-AKT,and p-mTOR(P<0.01).In MHCC97H cells,p-PI3K,p-AKT,and p-mTOR protein expression levels were significantly decreased(P<0.01)after adding GsMTx4 compared with the control group,and p-PI3K,p-AKT,and p-mTOR were significantly decreased(P<0.01)after transfecting siPiezol compared with the siCtrl group.8.In Hep3B and MHCC97H cells,cell proliferation ability was decreased in siPiezol group compared with siCtrl group(P<0.05);compared with siCtrl+3-MA group,cell proliferation ability was decreased in siPiezol+3-MA group(P<0.05);compared with siPiezol group,siPiezol+3-MA group The cell proliferation ability was increased in siPiezol+3-MA group compared with siPiezol group(P<0.05).9.In Hep3B and MHCC97H cells,P62 was significantly down-regulated and LC3-Ⅱwas significantly increased after transfection with siPiezol(P<0.05);P62 was significantly down-regulated and LC3-Ⅱ was significantly increased after adding 3-MA inhibitor to cells transfected with siCtrl(P<0.05);adding 3-MA inhibitor reversed the protein expression of P62 and LC3-Ⅱ,with P62 upregulated and LC3-Ⅱ downregulated(P<0.05).10.In Hep3B and MHCC97H cells,Bax,Cleaved Caspase 3 protein expression was significantly higher in the siPiezo1 group compared with the siCtrl group(P<0.05);compared with the siCtrl+3-MA group,Bax,Cleaved Caspase 3 protein expression in the siPiezol+3-MA group was significantly increased(P<0.05);Bax,Cleaved Caspase 3 protein expression was decreased in shPiezo1+3-MA group compared with siPiezo1 group(P<0.05).11.In Hep3B and MHCC97H cells,cell proliferation was decreased in siPiezol group compared with siCtrl group(P<0.05);cell proliferation ability was decreased in siPiezol+Rapa group compared with siCtrl+Rapa group(P<0.05);and siPiezol+Rapa group compared with siPiezol group.The cell proliferation ability was decreased in siPiezo1+Rapa group compared with siPiezo1 group(P<0.05).12.In Hep3B and MHCC97H cells,LC3-Ⅱ protein increased and P62 protein decreased after transfection with siPiezol(P<0.05);LC3-Ⅱ increased and P62 protein decreased after adding Rapa to cells transfected with siCtrl(P<0.05);LC3-Ⅱ increased and P62 protein decreased after adding Rapa to cells transfected with siPiezol(P<0.05);LC3-Ⅱ increased and P62 protein decreased after adding Rapa to cells transfected with siPiezol(P<0.05).Ⅱ was elevated and P62 protein was decreased in cells transfected with siPiezol(P<0.05).13.In Hep3B and MHCC97H cells,Bax and Cleaved Caspase 3 protein expression was significantly higher in the siPiezo1 group compared with the siCtrl group(P<0.01);compared with the siCtrl+Rapa group,Bax and Cleaved Caspase 3 protein expression in the siPiezol+Rapa group was significantly higher in the siPiezo1+Rapa group compared to the siCtrl+Rapa group(P<0.05);Bax,Cleaved Caspase 3 protein expression was higher in the siPiezol+Rapa group compared to the siPiezol group(P<0.05).14.Compared with liver tissues of mice with knockdown Piezol,mice without knockdown Piezo1 had more liver tumors and heavier tumor weight;higher AFP expression and more vacuoles were shown by HE staining.15.The tumors inoculated with transfected siPiezol liver cancer cells were smaller in size and lighter in tumor weight compared with those inoculated with transfected siCtrl liver cancer cells(P<0.05).Ⅱ.Potential mechanism of synergistic regulation of Piezol by JPHY drug serum and DOX and screening of effective active ingredients of JPHY1.The proliferation inhibition of Hep3B and MHCC97H cells was observed in a concentration-dependent manner by JPHY and DOX.The IC50 values of Hep3B and MHCC97H cells were reduced by the combination of JPHY and DOX.2.The results of plate clone formation assay,cell scratch assay and Transwell invasion assay showed that JPHY and DOX could inhibit the clone formation,migration and invasion ability of Hep3B and MHCC97H cells.The inhibition effect was most significant when JPHY was combined with DOX.The results showed that JPHY could synergize with DOX and enhance the inhibitory effect of DOX on the proliferation,migration and invasion ability of hepatocellular carcinoma cells.3.The results of apoptosis assay showed that JPHY and DOX could promote apoptosis in Hep3B and MHCC97H cells,and the number of apoptosis showed a more significant increase when JPHY combined with DOX than the DOX group.The results indicated that JPHY and DOX could enhance apoptosis of hepatocellular carcinoma cells under the effect of DOX.4.Systemic pharmacological analysis showed that there were 80 targets of DOX for hepatocellular carcinoma and 252 targets of JPHY for hepatocellular carcinoma;GO enrichment analysis of JPHY and DOX showed correlation with apoptosis and DNA damage,and KEGG enrichment analysis showed that both drugs focused on PI3K/AKT signaling pathway.5.Western Blot assay suggested that Piezo-1,p-PI3K,p-AKT,p-mTOR protein levels decreased in Hep3B and MHCC97H cells under the intervention of J JPHY and DOX(P<0.05),and the effect of JPHY combined with DOX in down-regulating protein expression was the most significant(P<0.01).6.The MDC method detected that JPHY and DOX promoted cellular autophagy in Hep3B cells and MHCC97H,and the effect of JPHY combined with DOX to promote autophagy was more significant than the effect of JPHY and DOX alone.Western Blot assay suggested that LC3-Ⅱ level was significantly increased and P62 level was significantly decreased under the effect of JPHY and DOX.The Western Blot assay showed that the LC3-Ⅱ level was significantly increased and the P62 level was significantly decreased under the effect of JPHY and DOX(P<0.05).7.The results of in vivo tumor suppression experiments of JPHY and DOX showed that JPHY had a significant inhibitory effect on subcutaneous graft tumor of MHCC97H cells(P<0.05),and the inhibitory effect of DOX compared with JPHY was significant(P<0.05),while the best tumor suppression effect was achieved when JPHY combined with DOX.The tumor HE staining results suggested that the combined drug had better cancer suppression effect and tumor tissue loosening than single drug under the intervention of JPHY and DOX.The tumor TUNEL staining results suggested that the combined drug promoted the apoptosis of tumor cells more than the single drug.8.HE staining of the organs of mice showed that there were no obvious lesions in the group of JPHY,DOX group and combined drug group,suggesting that the drugs did not cause damage to the organs.Meanwhile,the results of serum examination showed that the AST,ALT and CRE of JPHY group,DOX group and combined drug group were all within the normal range.9.The mass spectrometry analysis yielded 13 active ingredients,and the molecular docking was performed by combining the top 8 active compounds with the most therapeutic targets in systemic pharmacology.The 8 active compounds with the best binding capacity were selected.Conclusion1.Bioinformatics and molecular biology experiments revealed that Piezo1 is highly expressed in hepatocellular carcinoma.2.Piezo1 may mediate cellular autophagy and apoptosis through PI3K/AKT/mTOR pathway to inhibit the growth of hepatocellular carcinoma cells.3.Through systemic pharmacology and molecular biology,it was verified that JPHY synergized with DOX to inhibit the growth of hepatocellular carcinoma cells through Piezo1-regulated PI3K/AKT/mTOR pathway-mediated cell autophagy and apoptosis.4.Dioscin,Glycyrrhizic acid and other six active compounds were found to be the active components in JPHY with good binding ability to Piezo1 by mass spectrometry and molecular docking analysis. |
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